Purification and identification of cutinases from <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis> and <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.     

Role of plant lipid transfer proteins in plant cell physiology-a concise review

Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.     

Can electrons travel through actin microfilaments and generate oxidative stress in retinol treated Sertoli cell?

In early reports our research group has demonstrated that 7 μM retinol (vitamin A) treatment leads to many changes in Sertoli cell metabolism, such as up-regulation of antioxidant enzyme activities, increase in damage to biomolecules, abnormal cellular division, pre-neoplasic transformation, and cytoskeleton conformational changes. These effects were observed to be dependent on the production of reactive oxygen species (ROS), suggesting extra-nuclear (non-genomic) effects of retinol metabolism. Besides 7 μM retinol treatment causing oxidative stress, we have demonstrated that changes observed in cytoskeleton of Sertoli cells under these conditions were protective, and seem to be an adaptive phenomenon against a pro-oxidant environment resulting from retinol treatment. We have hypothesized that the cytoskeleton can conduct electrons through actin microfilaments, which would be a natural process necessary for cell homeostasis. In the present study we demonstrate results correlating retinol metabolism, actin architecture, mitochondria physiology and ROS, in order to demonstrate that the electron conduction through actin microfilaments might explain our results. We believe that electrons produced by retinol metabolism are dislocated through actin microfilaments to mitochondria, and are transferred to electron transport chain to produce water. When mitochondria capacity to receive electrons is overloaded, superoxide radical production is increased and the oxidative stress process starts. Our results suggested that actin cytoskeleton is essential to oxidative stress production induced by retinol treatment, and electrons conduction through actin microfilaments can be the key of this correlation.     

Aggregative adherence of uropathogenic Proteus mirabilis to cultured epithelial cells

Proteus mirabilis is an important cause of urinary tract infection (UTI) in patients with complicated urinary tracts. Thirty-five strains of P. mirabilis isolated from UTI were examined for the adherence capacity to epithelial cells. All isolates displayed the aggregative adherence (AA) to HEp-2 cells, a phenotype similarly presented in LLC-MK(2) cells. Biofilm formation on polystyrene was also observed in all strains. The mannose-resistant Proteus-like fimbriae (MR/P), Type I fimbriae and AAF/I, II and III fimbriae of enteroaggregative Escherichia coli were searched by the presence of their respective adhesin-encoding genes. Only the MR/P fimbrial subunits encoding genes mrpA and mrpH were detected in all isolates, as well as MR/P expression. A mutation in mrpA demonstrated that MR/P is involved in aggregative adherence to HEp-2 cells, as well as in biofilm formation. However, these phenotypes are multifactorial, because the mrpA mutation reduced but did not abolish both phenotypes. The present results reinforce the importance of MR/P as a virulence factor in P. mirabilis due to its association with AA and biofilm formation, which is an important step for the establishment of UTI in catheterized patients.     

The Burkholderia cepacia bceA gene encodes a protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities

The bceA gene is part of the Burkholderia cepacia IST408 exopolysaccharide (EPS) biosynthetic cluster. It encodes a 55.3-kDa bifunctional protein (type II PMI family) with phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. GMP activity is strongly dependent on the presence of Ca(2+) or Mn(2+), while PMI activity can use a broader variety of divalent cations (Ca(2+)>Mn(2+)>Mg(2+)>Co(2+)>Ni(2+)). The lack of a functional bceA gene does not affect EPS production yield in a non-polar insertion bceA mutant. The in silico search for putative bceA homologues revealed the presence of 2-5 bceA orthologues in the Burkholderia genomes available. This suggests that in B. cepacia IST408 putative bceA functional homologues may compensate the bceA mutation. However, the viscosity of aqueous solutions prepared with the EPS produced by the bceA mutant was significantly reduced compared with wild-type biopolymer and the mutant forms biofilms with a size reduced by 6-fold.     

Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.     

Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H₂O₂ to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T_m values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.